Pharmaceutical Composition Comprising Mixed Antibody of Anti-Ctla4 and Anti-Pd1 and Therapeutic Use Thereof

The present disclosure relates to the treatment of cancer, particularly non-small cell lung cancer, including immunotherapy and combination therapy. More specifically, the present disclosure relates to the use of a pharmaceutical composition comprising a mixture of antibodies of anti-CTLA4 and anti-PD1 for treating non-small cell lung cancer at stage II-IIIB after complete surgical resection, or locally advanced or metastatic non-small cell lung cancer that is negative for PD-L1 expression (PDL1 TPS<1%), has no epidermal growth factor receptor (EGFR)-sensitive mutation or altered anaplastic lymphoma kinase (ALK) gene translocation, and has not been subjected to a systemic anti-tumor therapy.

According to the data from GLOBOCAN in 2020, the incidence rate of lung cancer ranks second among cancers worldwide with 2.2 million cases, and its mortality rate ranks first with 1.8 million lung cancer deaths worldwide. In China, its incidence rate and mortality rate are in the first place, and new lung cancer cases and deaths are 816 thousand and 715 thousand, respectively.

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers, and includes two major pathological types, squamous cell carcinoma and non-squamous cell carcinoma. Non-squamous NSCLC accounts for about 60% of NSCLC, with lung adenocarcinoma being the most common type, accounting for about 40% of NSCLC. In lung adenocarcinoma, approximately 10% of Caucasian patients and 40-60% Asian patients carry EGFR sensitive mutations, mainly including a deletion in EGFR exon 19 and a mutation in L858R in exon 21. There are also mutations in genes such as ALK and ROS1.

About 30% of all NSCLC patients were diagnosed with resectable early NSCLC at initial diagnosis. Surgery is the primary treatment for patients with early NSCLC; and adjuvant chemotherapy is a common treatment for patients with surgically resectable locally advanced NSCLC. In the population receiving a post-operative adjuvant therapy, 20%-30% of patients who died within 5 years were at stage I, 50% at stage II, and 60% at stage IIIA. However, the 5-year survival rate decreases from 90% for stage IA1 of resection to 41% for stage IIIA, indicating micrometastasis in some patients at the time of surgical resection, resulting in treatment failure. At present, for immunotherapy of this part of the population, only Atezolizumab has been approved in China, with a low patient access. There is an urgent need for more kinds of effective medicines.

For patients without any genetic variations (including EGFR mutation or ALK gene rearrangement, also referred to as altered ALK gene translocation) in tumor, immune checkpoint inhibitors (ICIs) in combination with platinum-containing chemotherapy have become a first-line standard treatment. By taking studies including the Keynote-042 study together, it is shown from the data that the positive rate of PD-L1 expression is about 50˜60%, where only about ⅓ shows high expression, while patients with negative PD-L1 expression (defined as a tumor proportion score [TPS]<1%) account for about 40˜50% of NSCLC patients in China and are a broad treatment population.

Although immune checkpoint inhibitors in combination with platinum-containing chemotherapy have become the first-line standard treatment for advanced, driver-gene-negative NSCLC, there are significant differences in efficacy among populations with different PD-L1 expression levels, and in particular, there is lower efficacy in patients with negative expression of PD-L1. Under the same treatment regimen, the median PFS (mPFS) in patients with negative expression of PD-L1 was 3 months or more lower than that of patients with high PD-L1 expression (TPS>50%), and the median OS (mOS) was 6 months or more lower than that of patients with high PD-L1 expression (TPS>50%). For example, in the Keynote-189 study, patients with negative and high PD-L1 expression have mPFSs of 6.1 months and 9.4 months, respectively, and mOSs of 17.2 months and 27.7 months, respectively. NSCLC patients with negative PD-L1 expression and negative driver genes have a far unmet therapeutic need.

Established results suggest that dual immunotherapeutic drugs (combination of two immune checkpoint inhibitors) may have a survival benefit in tumor patients. However, dual immunotherapeutic drugs in combination with chemotherapy have not been approved in China at present. Immunotherapeutic drugs approved in China are used for locally advanced or metastatic NSCLC in the whole population or those with positive PD-L1 expression. Therefore, there is an urgent need for a large randomized and controlled study to validate the therapeutic efficacy of dual immunotherapeutic drugs in combination with chemotherapy on populations with PDL1-expression-negative, driver-gene-negative, locally advanced or advanced metastatic NSCLC.

ZPML265 is a pharmaceutical formulation of a mixture of antibodies composed of a recombinant humanized IgG1 monoclonal antibody targeting human CTLA4 and a recombinant humanized IgG4 monoclonal antibody targeting human PD1, the two different antibodies being produced by a single host cell. The mixture of antibodies specifically binds to CTLA4 and PD1 at the same time, thereby blocking two immune checkpoint signaling pathways relating to CTLA4 and B7-1/B7-2 as well as PD-1 and PD-L1, relieving the inhibitory effects of the two pathways on T lymphocytes, restoring their functional activity and anti-tumor immune response, which in turn achieve the goal of combatting and killing tumor in the body.

Therefore, there is an unmet clinical need among patients with stage II-IIIB non-small cell lung cancer after complete surgical resection and PDL1-expression-negative, driver-gene-negative, locally advanced or metastatic non-small cell lung cancer. There is an urgent need to develop more effective medicines.

The technical problem to be solved by the present disclosure is to provide an immunotherapy to fill the clinical gap of no dual-target immunotherapy drugs for non-small cell lung cancer in China, thus addressing this unmet clinical need.

The present disclosure provides a pharmaceutical composition comprising an effective amount of a mixture of antibodies of anti-CTLA4 and anti-PD1 for treating non-small cell lung cancer.

Meanwhile, the present disclosure also provides a method for treating non-small cell lung cancer using a pharmaceutical composition comprising an effective amount of a mixture of antibodies against CTLA4 and PD1.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, which includes, but is not limited to, a chemotherapeutic agent (chemotherapeutic drug), a cytotoxic agent, a radiotherapeutic agent, a cancer vaccine, a cytoreductive agent, a targeted anticancer agent, an anti-angiogenic agent, a biological response modifier, a cytokine, a hormone, an anti-metastatic agent, and an immunotherapeutic agent.

In some embodiments, the additional therapeutic agent is a chemotherapeutic drug. Preferred chemotherapeutic drug is carboplatin/cisplatin, paclitaxel/vinorelbine, and/or pemetrexed/vinorelbine, wherein carboplatin and cisplatin may be used interchangeably and alternatively, paclitaxel and vinorelbine may be used interchangeably and alternatively, and pemetrexed and vinorelbine may be used interchangeably and alternatively.

In some embodiments, the non-small cell lung cancer described in the present disclosure is at stage III or IV according to the TNM staging.

In some other embodiments, the non-small cell lung cancer is non-small cell lung cancer at stage II-IIIB after complete surgical resection.

In some other embodiments, the non-small cell lung cancer is locally advanced or metastatic non-small cell lung cancer that is negative for PD-L1 expression, has no epidermal growth factor receptor (EGFR)-sensitive mutation or altered anaplastic lymphoma kinase (ALK) gene translocation, and has not been subjected to a systemic anti-tumor therapy.

In the present disclosure, the non-small cell lung cancer comprises squamous non-small cell lung cancer and non-squamous non-small cell lung cancer.

Preferably, when the non-small cell lung cancer is squamous non-small cell lung cancer, the mixture of antibodies is administered at a dose of 5 mg/kg by intravenous infusion on day 1 of each cycle, with one treatment cycle lasting 21 days. In addition, additional chemotherapeutic drugs, which are carboplatin/cisplatin and paclitaxel/vinorelbine, may be administered. Carboplatin is at a dose of 5 mg/mL/min based on an area under the curve (AUC), or cisplatin is at a dose of 75 mg/m2; and paclitaxel is at a dose of 175 mg/m2, or vinorelbine is at a dose of 25 mg/m2; administration is performed by intravenous infusion on day 1 of each cycle, with one treatment cycle lasting 21 days; and if vinorelbine is selected, vinorelbine is administered at 25 mg/magain on day 8 of an administration cycle. Further, after the completion of 2 to 4 administration cycles of the mixture of antibodies and the chemotherapeutic drugs, the mixture of antibodies is used for maintenance treatment at a dose of 5 mg/kg once every three weeks by intravenous infusion.

Preferably, when the non-small cell lung cancer is non-squamous non-small cell lung cancer, the mixture of antibodies is administered at a dose of 5 mg/kg by intravenous infusion on day 1 of each cycle, with one treatment cycle lasting 21 days. In addition, additional chemotherapeutic drugs, which are carboplatin/cisplatin and pemetrexed/vinorelbine, may be administered. Carboplatin is at a dose of 5 mg/mL/min based on an area under the curve (AUC), or cisplatin is at a dose of 75 mg/m; and pemetrexed is at a dose of 500 mg/m, or vinorelbine is at a dose of 25 mg/m; administration is performed by intravenous infusion on day 1 of each cycle, with one treatment cycle lasting 21 days; and if vinorelbine is selected, vinorelbine is administered at 25 mg/magain on day 8 of an administration cycle. Further, after the completion of 2 to 4 administration cycles of the mixture of antibodies and the chemotherapeutic drugs, the mixture of antibodies is used for maintenance treatment at a dose of 5 mg/kg once every three weeks by intravenous infusion.

In some embodiments, the mixture of antibodies is produced by a single host cell comprising nucleic acids encoding two different antibodies, i.e. both a nucleic acid encoding an anti-CTLA4 antibody and a nucleic acid encoding an anti-PD1 antibody, wherein the sequences of heavy chain HCDR1, HCDR2 and HCDR3 of the anti-CTLA4 antibody are set forth in SEQ ID NOs: 1, 2 and 3, respectively; the sequences of light chain LCDR1, LCDR2 and LCDR3 of the anti-CTLA4 antibody are set forth in SEQ ID NOs: 4, 5 and 6, respectively; the sequences of heavy chain HCDR1, HCDR2 and HCDR3 of the anti-PD1 antibody are set forth in SEQ ID NOs: 9, 10 and 11, respectively; and the sequences of light chain LCDR1, LCDR2 and LCDR3 of the anti-PD1 antibody are set forth in SEQ ID NOs: 12, 13 and 14, respectively.

In some embodiments, the sequence of heavy chain variable region of the anti-CTLA4 antibody is set forth in SEQ ID NO:7, the sequence of light chain variable region of the anti-CTLA4 antibody is set forth in SEQ ID NO:8, the sequence of heavy chain variable region of the anti-PD1 antibody is set forth in SEQ ID NO: 15, and the sequence of light chain variable region of the anti-PD1 antibody is set forth in SEQ ID NO: 16.

In some embodiments, the sequence of heavy chain of the anti-CTLA4 antibody is set forth in SEQ ID NO: 17, the sequence of light chain of the anti-CTLA4 antibody is set forth in SEQ ID NO: 18, the sequence of heavy chain of the anti-PD1 antibody is set forth in SEQ ID NO: 19, and the sequence of light chain of the anti-PD1 antibody is set forth in SEQ ID NO:20.

Meanwhile, the present disclosure also provides the use of the mixture of antibodies comprising an effective amount of anti-CTLA4 antibody and anti-PD1 antibody and a chemotherapeutic drug in the manufacture of a pharmaceutical composition for treating stage II-IIIB non-small cell lung cancer after complete surgical resection.

In addition, the present disclosure also provides the use of the mixture of antibodies comprising an effective amount of anti-CTLA4 antibody and anti-PD1 antibody and a chemotherapeutic drug in the manufacture of a pharmaceutical composition for treating locally advanced or metastatic non-small cell lung cancer that is negative for PD-L1 expression, has no epidermal growth factor receptor (EGFR)-sensitive mutation or altered anaplastic lymphoma kinase (ALK) gene translocation, and has not been subjected to a systemic anti-tumor therapy.

Results from a phase I clinical study of the mixture of antibodies of the present disclosure as a monotherapy have shown that there are exact curative effects and adequate safety on the enrolled patients with non-small cell lung cancer. Further, clinical studies that have been conducted based on the mixture of antibodies of the present disclosure in combination with chemotherapy in non-small cell lung cancer have also shown a synergistic anti-tumor effect, exhibiting superior clinical efficacy. Therefore, the mixture of antibodies as a monotherapy, or a combined regimen of the mixture of antibodies+carboplatin/cisplatin+paclitaxel/vinorelbine, or of the mixture of antibodies+carboplatin/cisplatin+pemetrexed/vinorelbine provided in the present disclosure can be used for medical treatment of stage III or IV non-small cell lung cancer, is expected to be controllable in safety and to have good patient compliance, and can largely fill the blank where there is no dual-target immunotherapy drug approved for non-small cell lung cancer in China, thereby solving the unmet urgent clinical need.

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application had been specifically and individually indicated to be incorporated by reference.

Before the present disclosure is described in detail below, it is to be understood that the present disclosure is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It is also to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise indicated, it is to be understood that the numerical ranges or the manners described in terms of ranges are for purposes of brevity and convenience only, and are not to be considered as a strict limitation on the scope of the present disclosure. Accordingly, the description in terms of ranges should be considered to specifically disclose all possible sub-ranges and all possible specific numerical points within that range, as if such sub-ranges and numerical points had been expressly written herein. The above principles apply equally regardless of the width of the numerical values. When description is made by using a range, the range includes the endpoints of the range.

In the cases relating to a measurable value, such as an amount and a temporary time duration, the term “about” refers to a change by ±20%, in certain cases by ±10%, in certain cases by ±5%, in certain cases ±by 1%, or in certain cases ±by 0.1%, of a specified value.

As used herein, three-letter codes and single-letter codes for amino acids are described in J. Biol. Chem, 243, p3558 (1968).

As used herein, the term “antibody” typically refers to a Y-shaped tetrameric protein containing two heavy (H) polypeptide chains (HC) and two light (L) polypeptide chains (LC) held together by covalent disulfide bonds and non-covalent interactions. A natural IgG antibody has such structure. Each light chain contains a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant domain (CH) (also known as heavy chain constant region (CH)).

Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG and IgM, their corresponding heavy chain constant domains being referred to as a, 8, ¿, y and u, respectively. IgG and IgA can be further divided into different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3 and IgG4; and IgA can be divided into IgA1 and IgA2. A light chain of an antibody from any vertebrate species may be assigned to one of two apparently distinct types, termed K and 2, based on the amino acid sequence of its constant domain.

The term “variable region” or “variable domain” presents significant changes in amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable regions of each light/heavy chain pair form an antigen binding site such that an intact IgG antibody has two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVR), or more generally, as complementarity determining regions (CDRs). VH and VL each having four backbone regions, FRs (or framework regions), denoted by FR1, FR2, FR3 and FR4, respectively. Thus, CDR and FR sequences typically occur in the following sequence of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

Types of “antibodies” in a broad sense may include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotype antibodies, synthetic antibodies (including muteins and variants thereof), and the like.

The term “monoclonal antibody” refers to an antibody that is produced by a single cell clone and is substantially homogeneous, targeting only one particular epitope. Monoclonal antibody can be prepared using a variety of techniques known in the art, including hybridoma techniques, recombination techniques, phage display techniques, those with transgenic animals, synthetic techniques, or combinations of the foregoing, and the like.

It should be noted that the division of the CDRs and FRs of an antibody variable region of the present disclosure is determined according to the Kabat definition. Other naming and numbering systems, such as Chothia, IMGT or AHo, are also known to those skilled in the art. Thus, humanized antibodies comprising one or more CDRs derived from any naming system, based on the antibody sequences of the present disclosure, are expressly maintained within the scope of the present disclosure.

The term “antigen” refers to a substance that is recognized and specifically bound by an antibody or antibody binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, single domain, multiple domains, or intact extracellular domain (ECD) or protein.

The term “epitope” refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. The epitope may be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed due to tertiary folding of a protein.

The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear, cyclic or branched. It may contain a modified amino acid, in particular a conservatively modified amino acid, and it may be interrupted by a non-amino acid. The terms also include, for example, an amino acid polymer that has been modified by glycosylation, lipidation, acetylation, phosphorylation, methylation, and the like.

As used herein, the term “mixture of antibodies” refers to that containing a limited number, optionally no more than two, three, four, five, six, seven, eight, nine or ten major antibody species produced from a host cell (optionally a cell from a single host cell line) that has been transfected with DNAs encoding at least two different antibodies (optionally full-length primate IgG antibodies) having different binding specificities. In some embodiments, DNAs encoding at least two different heavy chains (HC) and at least two different light chains (LC) may be introduced into the same host cell, for example, the host cell may be transfected with DNAs encoding at least two but no more than four different antibodies with different binding specificities. In some embodiments, the sequences of all transfected DNAs encoding HC and LC may be mutated, thereby altering the amino acid sequence of the antibody such that non-homologous HC/LC pairing is not facilitated, while homologous HC/LC pairing is highly facilitated. Where two different HCs are introduced into a host cell, one or both of the two different HCs may optionally be altered so that the formation of heterodimers is not facilitated. In some embodiments, only one heavy chain is altered to prevent the formation of heterodimers. In some embodiments, where DNAs encoding only two different antibodies are introduced into a host cell, only one of the antibodies encoded by the DNAs comprises one or more partner-directed alterations such that homologous HC/LC pairing is facilitated, while the other antibody does not comprise such alterations. In some embodiments, the host cell produces only two major antibody species, where each HC primarily paired with its homologous LC, and most antibodies are tetramers containing two heavy chains having the same amino acid sequence and two light chains having the same amino acid sequence (see PCT/US2017/030676).

The term “pharmaceutical composition” refers to a formulation or combination of formulations that comprises one, two or more active ingredients, allows the active ingredients contained therein to exist in a biologically effective form, and does not include additional ingredients with unacceptable toxicity to a subject to whom the formulation is administered. When the “pharmaceutical composition” is in the form of a combination of separate formulations containing two or more different active ingredients, they may be administered simultaneously, sequentially, separately or at intervals, the purpose of which is to exert biological activities of the various active ingredients, together for the treatment of the disease(s).

The term “pharmaceutical carrier” or “pharmaceutically acceptable carrier” refers to a diluent, an adjuvant (e.g., Freund's adjuvant (complete and incomplete)), an excipient, or a vehicle administered with a therapeutic agent.

The term “effective amount” refers to a dosage of a pharmaceutical formulation comprising the active ingredients of the present disclosure that, upon administration to a patient in a single dose or multiple doses, produces desired effects in the patient being treated. An effective amount can be easily determined by an attending physician as a person skilled in the art by considering a variety of factors such as race differences; weight, age, and health status; specific disease involved; severity of the disease; response of an individual patient; specific antibodies administered; administration mode; bioavailability profile of the administrated formulation; selected dosing regimen; and use of any concomitant therapy.

The terms “host cell”, “host cell line” and “host cell culture” may be used interchangeably and refer to a cell with an exogenous nucleic acid introduced therein, including a progeny of such cell. Host cell includes a “transformant” and a “transformed cell”, which includes an initially transformed cell and a progeny originated therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parent cell and may contain mutation(s). Included herein are mutated progenies having the same function or biological activity as those screened or selected from the initially transformed cell.

As used herein, the term “transfection” refers to the introduction of an exogenous nucleic acid into a eukaryotic cell. Transfection can be accomplished by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.

The term “isolated polynucleotide” or “isolated nucleic acid” refers to a nucleic acid molecule, DNA or RNA that has been removed from its native environment. For example, for the purposes of the present disclosure, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of an isolated polynucleotide include a recombinant polynucleotide maintained in a heterologous host cell or a (partially or substantially) purified polynucleotide in a solution. An isolated polynucleotide includes a polynucleotide molecule contained in a cell typically containing the polynucleotide molecule, but the polynucleotide molecule is present outside the chromosome or at a chromosomal location different from its native chromosomal location. An isolated RNA molecule includes an in vivo or in vitro RNA transcript of the present disclosure, as well as its positive- and negative-stranded forms and double-stranded forms. An isolated polynucleotide or nucleic acid of the present disclosure also includes such molecule produced synthetically. In addition, the polynucleotide or nucleic acid may or may not include a regulatory element such as a promoter, a ribosome binding site, or a transcription terminator.

The terms “nucleic acid molecule code”, “encoding DNA sequence” and “encoding DNA” refer to the order of deoxyribonucleotides along the deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of the amino acids along the polypeptide (protein) chain. Thus, the nucleic acid sequence encodes an amino acid sequence.

Methods for producing and purifying an antibody and an antigen-binding fragment are well known and can be found in the prior art, such as in the Antibodies: A Laboratory Manual, Chapters 5-8 and 15, by Cold Spring Harbor. The engineered antibodies or antigen-binding fragments thereof in the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains may be cloned and recombined into an expression vector. The recombinant vector for immunoglobulin expression may be stably transfected into CHO cells. A stable clone is obtained by expressing antibodies that specifically bind to human antigens. The positive clone is expanded in a serum-free medium in a bioreactor to produce the antibodies. The culture medium into which the antibodies are secreted may be purified and collected by conventional techniques. The antibodies may be filtrated and concentrated using conventional methods. Soluble mixtures and multimers may also be removed by conventional methods, such as molecular sieves and ion exchange.